Treatment of chronic inflammatory conditions

ABSTRACT

A method for the treatment of a chronic inflammatory condition in a patient which comprises administration to the patient of an agent which blocks or inhibits IL-3 signalling events in the patient.

FIELD OF THE INVENTION

This invention relates generally to the treatment of chronicinflammatory conditions, and in particular to a method for reducing orotherwise ameliorating the effects of such conditions in patients. Inone particular embodiment, the present invention relates to thetreatment of arthritis, particularly rheumatoid arthritis which is achronic, inflammatory disease characterised by the presence of numerousinflammatory mediators and by the destruction of diarthrodial joints.

BACKGROUND OF THE INVENTION

Interleukin-3 (IL-3) is a cytokine that can improve the body's naturalresponse to disease as part of the immune system. IL-3 stimulates thedifferentiation of multipotent hematopoietic stem cells (pluripotent)into myeloid progenitor cells as well as stimulating proliferation ofall cells in the myeloid lineage (erythrocytes, thrombocytes,granulocytes, monocytes, and dendritic cells). It is secreted byactivated T cells to support growth and differentiation of T cells fromthe bone marrow in an immune response.

IL-3 exerts its activity through binding to a specific cell surfacereceptor known as the Interleukin-3 receptor (IL-3 R). IL-3 R is aheterodimeric structure composed of a 70 kDa IL-3 R alpha (CD123) and a120-140 kDa IL-3 R beta (CD131). The IL-3 R alpha chain (IL-3Rα) has avery short intracellular domain while the IL-3 R beta chain (IL-3Rβ) hasa very large cytoplasmic domain. IL-3 R alpha binds IL-3 with relativelylow affinity. In the presence of IL-3 R beta, however, IL-3 R alpha hasa much higher affinity for IL-3. It is not clear how signal transductionoccurs following IL-3 binding, however recent studies suggest signallingrequires formation of a higher order complex comprising a dodecamer ¹.The IL-3 R beta chain is also shared by the receptors for IL-5 andGM-CSF. Cells known to express IL-3 receptors include hematopoieticprogenitors, mast cells, basophils and blood monocytes as well as moremature cells of various hematopoietic lineages including monocytes,macrophages, neutrophils, basophils, mast cells, eosinophils,megakaryocytes, erythroid cells, and CD5⁺ B cell sub-populations ².Non-hematopoietic cells have also been shown to express the receptorincluding some endothelial cells, stromal cells, dendritic cells andLeydig cells ^(2,3).

IL-3 provides a potentially important connection between the immune andhemopoietic systems ⁴. It may be important for the production andfunction of mast cells and basophils particularly during immunereactions ⁵. IL-3 can also promote the growth and activation ofmacrophage lineage populations⁶⁻⁸ and can help to generate dendriticcells ⁹. As noted above, IL-3 signalling is mediated by a commonreceptor beta-subunit (IL-3Rβ) and a specific ligand-bindingalpha-subunit (IL-3Rαa), although in the mouse there is an additionalbeta-subunit ¹⁰.

Very little is known regarding the role of IL-3 in chronic inflammatoryconditions, such as rheumatoid arthritis (RA). IL-3 mRNA could not bedetected in the synovium of

RA patients in one study ¹¹ but was found in a later study ¹²; some butnot all RA patients have been found to have detectable IL-3 in thecirculation¹³ and there is an association between a single-nucleotidepolymorphism in the IL-3 gene promoter and RA¹⁴. However, IL-3 levelsdecrease during arthritis progression in a rat arthritis model¹⁵ and ithas been reported recently that IL-3 administration inhibits murineinflammatory arthritis¹⁶.

In work leading to the present invention, the inventors have found thatthe effects of chronic inflammatory conditions such as RA can beinhibited or reduced by blocking or interfering with the ligand/receptorinteraction between IL-3 and IL-3R. This finding is quite surprising andwas unexpected in view of the recent report¹⁶ suggesting that IL-3administration has the potential to diminish the inflammatory responseand indirectly arrest cartilage and bone loss in inflammatory arthritis.

Bibliographic details of the publications referred to in thisspecification are referenced at the end of the description.

The reference in this specification to any prior publication (orinformation derived from it), or to any matter which is known, is not,and should not be taken as an acknowledgment or admission or any form ofsuggestion that that prior publication (or information derived from it)or known matter forms part of the common general knowledge in the fieldof endeavour to which this specification relates.

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof a stated integer or step or group of integers or steps but not theexclusion of any other integer or step or group of integers or steps.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a method for the treatmentof a chronic inflammatory condition in a patient, which comprisesadministration to the patient of an agent which blocks or inhibits IL-3signalling events in the patient.

In another aspect, the invention provides the use of an agent whichblocks or inhibits IL-3 signalling events in, or in the manufacture of amedicament for, the treatment of a chronic inflammatory condition in apatient.

In yet another aspect, the invention provides an agent for the treatmentof a chronic inflammatory condition in a patient, wherein said agentblocks or inhibits IL-3 signalling events in the patient.

In other aspects of this invention, the agent may be formulated in apharmaceutical composition together with one or more pharmaceuticallyacceptable excipients and/or diluents, or it may be provided in a kitwhich optionally includes instructions to use the agent in accordancewith a method for the treatment of a chronic inflammatory condition in apatient.

The chronic inflammatory condition may be, for example, arthritis, moreparticularly inflammatory arthritis such as RA.

Preferably, the agent is one which blocks or inhibits IL-3/IL-3R orIL-3/IL-3R alpha interactions in the patient. Preferably also thepatient is a human.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows collagen-induced arthritis (CIA) progression (clinicalscore) in mice receiving anti-IL-3 mAb or PBS (control). Resultsexpressed as mean±SEM. n=10 mice treated group, n=6 mice control group.

FIG. 2 shows effect of IL-3 signalling on the cell viability ofmonocytes (CD14+ derived from PBMC). The monocytes were plated at adensity of ˜1.8×10⁶ cells per 6 cm IWAKI (low adherence) TC dish andcultured for 7 days in RPMI+10% FCS and: IL-3 (3 ng/ml) alone, IL-3 (3ng/ml)+IL-3-R antibody, or IL-3 (0.3 ng/ml) alone, or IL-3 (0.3ng/ml)+IL-3-R antibody. IL-3-R antibody was used at 1 μg/ml in eachcase. At day 4 new IL-3 and IL-3-R antibodies were added. On day 7 cellswere removed and counted.

FIG. 3 shows IL-3 induces the basophil activation marker CD203c in adose-dependent manner. PBMC from urticaria patients (URT) and a normaldonor (NOR) were isolated and were stimulated with polyclonal IgE, FMLP,FMLP and IL-3 or an increasing concentration of IL-3. The percentage ofCD203+ve basophils were calculated after staining with an antibodycocktail to identify basophils and with an antibody against the basophilactivation marker CD203c then analyzing by flow cytometry.

FIG. 4 shows anti-IL-3R antibody blocks IL-3-induced basophilactivation. PBMC from a normal donor were isolated and stimulated withan increasing concentration of IL-3 in the presence or absence of aneutralizing anti-IL-3R antibody (CSL360). The percentage of CD203+vebasophils were calculated after staining with an antibody cocktail toidentify basophils and with an antibody against the basophil activationmarker CD203c then analyzing by flow cytometry.

FIG. 5 shows anti-CD123 monoclonal antibody (CSL362) depletes basophilin a time-dependent manner. PBMC from a normal donor were isolated andincubated without antibody or with a depleting anti-CD123 antibody(CSL362) for various times (as indicated). The percentage of basophilsremaining were calculated after staining with an antibody cocktail toidentify basophils and analyzing by flow cytometry.

FIG. 6 shows anti-CD123 mAb reproducibly depletes basophils within 24hr.

PBMC from three normal donors were isolated and incubated withoutantibody or with a depleting anti-CD123 antibody (CSL362) for 24 h. Thepercentage of basophils remaining were calculated after staining with anantibody cocktail to identify basophils and analyzing by flow cytometry.

FIG. 7 shows anti-CD123 mAb activates NK cells within 24 hr. PBMC fromthree normal donors were isolated and incubated without antibody (solidline) or with a depleting anti-CD 123 antibody (CSL362, dashed line) for24 h. Activation of NK cells (CD56+ve cells) was determined by CD16down-regulation. PBMC were stained with anti-CD56 antibodies to identifyNK cells and with anti-CD16 antibodies. Loss of CD16 staining, ascompared with the no antibody control, was determined by flow cytometry.

FIG. 8 shows depletion of murine basophils in peripheral blood by invivo administration of anti-IL-3Rα antibodies. BALB/c mice wereintravenously administered with an anti-IL-3Rα antibody (1C2),anti-CD200R3 (Ba103) or the isotype control antibodies mouse IgG2a(mIgG2a) or rat IgG2b (rIgG2b). All antibodies were injected at 30 μgper mouse except for 1C2 which was injected at 18 μg per mouse.Peripheral blood cells were isolated 24 h post antibody administrationand stained for FcεR1α and CD49b expression to identify basophils.Representative staining profiles from flow cytometry analysis are shown(a). Basophil populations are boxed. The percentage of basophils permouse were calculated from flow cytometry analysis and the mean (+SEM)of 3 mice per group are shown (b). ***: p<0.001, **: p<0.01.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect, the present invention provides a method for the treatmentof a chronic inflammatory condition in a patient, which comprisesadministration to the patient of an agent which blocks or inhibits IL-3signalling events in the patient. Preferably, the agent is one whichblocks or inhibits IL-3/IL-3R or IL-3/IL-3R alpha interactions in thepatient.

Chronic inflammatory conditions which may be treated in accordance withthe present invention are well known to persons skilled in this field,and include in particular arthritis, more particularly inflammatoryarthritis such as adult and juvenile RA. Other indications include butare not limited to chronic obstructive pulmonary disease (COPD);inflammatory bowel diseases (IBD) such as Crohn's disease and ulcerativecolitis; chronic inflammatory demyelinating polyneuropathy (CIDP);atherosclerosis; scleroderma; systemic lupus erythematosus (SLE);Sjogren's syndrome; gout; osteoarthritis; polymyalgia rheumatica;seronegative spondyloarthropathies including ankylosing spondylitis;Reiter's disease, psoriatic arthritis, mixed connective tissue disease(MCTD);

chronic Lyme arthritis; Still's disease; chronic urticaria; uveitisassociated with rheumatoid arthritis and disorders resulting ininflammation of the voluntary muscle and other muscles, includingdermatomyositis, inclusion body myositis, polymyositis, andlymphangioleiomyomatosis.

Reference herein to “treatment” is to be considered in its broadestcontext and includes both therapeutic treatment and prophylactic orpreventative measures. Patients in need of treatment include thosealready afflicted with a chronic inflammatory condition as well as thosein which such a condition is to be prevented. Patients who are partiallyor totally recovered from the condition might also be in need oftreatment. The term “treatment” does not necessarily imply that apatient is treated until total recovery. Accordingly, treatment includesreduction or amelioration of the symptoms of a particular chronicinflammatory condition as well as halting or at least retarding theonset, development or progress of, reducing the severity of, oreliminating, a particular chronic inflammatory condition.

The agent which is administered in accordance with the present inventionblocks or inhibits the activation of IL-3 signalling events in thepatient, preferably by blocking or inhibiting IL-3/IL-3R or IL-3/IL-3Ralpha interactions. As used herein, a reference to “blocks or inhibitsIL-3 signalling events in the patient” encompasses any interventionwhich leads to a decreased level of IL-3 initiated signalling. Suchinterventions include, by way of example, the use of agents whichspecifically block or inhibit the activation of IL-3 signalling events(e.g. agents which target IL-3, IL-3Rα, IL-3Rβ), as well as the use ofagents designated to selectively target cells capable of IL-3 signallingand by such targeting induce cell death (e.g. agents which target IL-3Rand carry an anti-cellular moiety).

In one embodiment of the invention, the agent may be an antigen bindingmolecule which binds selectively to IL-3, or to IL-3R, the IL-3R alphaor the IL-3R beta.

As used herein the term “antigen binding molecule” refers to an intactimmunoglobulin, including monoclonal antibodies, such as chimeric,humanized or human monoclonal antibodies, or to antigen-binding(including, for example, Fv, Fab, Fab′ and F(ab′)₂ fragments) and/orvariable-domain-comprising fragments of an immunoglobulin that competewith the intact immunoglobulin for specific binding to the bindingpartner of the immunoglobulin, e.g. a host cell protein. Regardless ofstructure, the antigen-binding fragments bind with the same antigen thatis recognized by the intact immunoglobulin. Antigen-binding fragmentsmay be produced synthetically or by enzymatic or chemical cleavage ofintact immunoglobulins or they may be genetically engineered byrecombinant DNA techniques. The methods of production of antigen bindingmolecules and fragments thereof are well known in the art and aredescribed, for example, in Antibodies, A Laboratory Manual, Edited by E.Harlow and D. Lane (1988), Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y., which is incorporated herein by reference.

Preferably, the antigen binding molecule is a monoclonal antibody.

In this embodiment of the invention, the antigen binding molecule maycomprise a modified Fc region, more particularly a Fc region which hasbeen modified to provide enhanced effector functions, such as enhancedbinding affinity to Fc receptors, antibody-dependent cell-mediatedcytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis(ADCP) and complement-dependent cytotoxicity (CDC). For the IgG class ofantibodies, these effector functions are governed by engagement of theFc region with a family of receptors referred to as the Fcγ receptors(FcγRs) which are expressed on a variety of immune cells. Formation ofthe Fc/FcγR complex recruits these cells to sites of bound antigen,typically resulting in signalling and subsequent immune responses.Methods for optimizing the binding affinity of the FcγRs to the antibodyFc region in order to enhance the effector functions, in particular toalter the ADCC and/or CDC activity relative to the “parent” Fc region,are well known to persons skilled in the art, and are described, forexample, in International Patent Publication No. WO 2009/070844. Thesemethods can include modification of the Fc region of the antibody toenhance its interaction with relevant Fc receptors and increase itspotential to facilitate ADCC and ADCP. Enhancements in ADCC activityhave also been described following the modification of theoligosaccharide covalently attached to IgG1 antibodies at the conservedAsn²⁹⁷ in the Fc region.

The term “binds selectively”, as used herein, in reference to theinteraction of an antigen binding molecule, e.g. an antibody or antibodyfragment, and its binding partner, e.g. an antigen, means that theinteraction is dependent upon the presence of a particular structure,e.g. an antigenic determinant or epitope, on the binding partner. Inother words, the antibody or antibody fragment preferentially binds orrecognizes the binding partner even when the binding partner is presentin a mixture of other molecules or organisms.

Without wishing to be bound by any particular theory, it is believedthat in this embodiment of the invention, by binding selectively toIL-3, or to IL-3R, IL-3R alpha or IL-3R beta, an antigen bindingmolecule such as an antibody or antibody fragment blocks or inhibits theligand/receptor interaction and thereby interferes with IL-3 signalactivation.

In one embodiment, the antigen binding molecule may be a monoclonalantibody which binds selectively to IL-3R alpha (CD123). Thus, theantigen binding molecule may be monoclonal antibody (MAb) 7G3, raisedagainst CD123, which has previously been shown to inhibit IL-3 mediatedproliferation and activation of both leukaemic cell lines and primarycells (see U.S. Pat. No. 6,177,678 to Lopez). Alternatively, the agentmay be the monoclonal antibody CSL360, a chimeric antibody obtained bygrafting the light variable and heavy variable regions of the mousemonoclonal antibody 7G3 onto a human IgG1 constant region (seeInternational Patent Publication No. WO 2009/070844). Like 7G3, CSL360binds to CD123(human IL-3Rα) with high affinity, competes with IL-3 forbinding to the receptor and blocks its biological activities. CSL360also has the advantage of potential utility as a human therapeutic agentby virtue of its human IgG1 Fc region which would be able to initiateeffector activity in a human setting. Moreover, it is likely that inhumans it would show reduced clearance relative to the mouse 7G3equivalent and be less likely to be immunogenic. Further examples ofthis antigen binding molecule include humanised antibody variants of 7G3or CSL360, fully human anti-CD123 antibodies and anti-CD123 antibodieswith enhanced effector function (such as ADCC activity) as described,for example in Example 4 of International Patent Publication No.WO2009/070844.

In another embodiment of the present invention, the agent may be an IL-3mutein which binds to IL-3R but either does not lead to or at leastresults in reduced IL-3 signal activation. Generally, these ‘IL-3muteins’ include natural or artificial mutants differing by theaddition, deletion and/or substitution of one or more contiguous ornon-contiguous amino acid residues. An example of an IL-3 mutein whichbinds to IL-3R but exhibits reduced IL-3 signal activation is a 16/84C→A mutant¹⁷. IL-3 muteins may also include modified polypeptides inwhich one or more residues are modified to, for example, increase theirin vivo half life. This could be achieved by attaching other elementssuch as a PEG group. Methods for the PEGylation of polypeptides are wellknown in the art.

In another embodiment of the present invention, the agent is a solublereceptor which is capable of binding to IL-3. Examples of such solublereceptors include the extracellular portion of IL-3R alpha or a fusionprotein comprising the extracellular portion of IL-3R alpha fused to theextracellular portion of IL-3R beta.

In yet another embodiment, the agent may comprise an anti-cellularmoiety that can be targeted to cells capable of IL-3 signalling toinduce cell death. In some embodiments, the agent may comprise theanti-cellular moiety conjugated to an antigen binding molecule whichbinds selectively to IL-3, or to IL-3R, IL-3R alpha or IL-3R beta. Inother embodiments the agent may comprise an anticellular moietyconjugated to a IL-3 mutein. Examples of suitable anti-cellular moietiesinclude chemotherapeutics, radioisotopes or cytotoxins.Chemotherapeutics include a hormone such as a steroid; ananti-metabolite such as cytosine arabinoside, fluorouracil, methotrexateor aminopterin; an anthracycline ; mitomycin C; a vinca alkaloid;demecolcine; etoposide; mithramycin; calicheamycin, CC-1065 andderivatives thereof, or an alkylating agent such as chlorambucil ormelphalan, a coagulant, a cytokine, growth factor, bacterial endotoxinor the lipid A moiety of bacterial endotoxin. Radioisotopes includeα-emitters such as, for example, 211 Astatine, 212Bismuth and213Bismuth, as well as β-emitters such as, for example, 131Iodine,90Yttrium, 177Lutetium, 153Samarium and 109Palladium, and Auger emitterssuch as, for example, 111Indium. Cytotoxins include generally a plant-,fungus-or bacteria-derived toxin, such as an A chain toxin, a ribosomeinactivating protein, a-sarcin, aspergillin, restirictocin, aribonuclease, diphtheria toxin or pseudomonas exotoxin, to mention justa few examples, as well as cytotoxins derived from marine organisms suchas sponges, such as Kahalalide F, Ecteinascidin (Yondelis™), or VariolinB, for example.

The agent is administered in an effective amount. An “effective amount”means an amount necessary at least partly to attain the desired responseor to delay or inhibit progression or halt altogether, the progressionof the particular condition being treated. The amount varies dependingupon the health and physical condition of the individual to be treated,the racial background of the individual to be treated, the degree ofprotection desired, the formulation of the composition, the assessmentof the medical situation, and other relevant factors. It is expectedthat the amount will fall in a relatively broad range that can bedetermined through routine trials. If necessary, the administration ofthe agent may be repeated one or several times. The actual amountadministered will be determined both by the nature of the conditionwhich is being treated and by the rate at which the agent is beingadministered.

Preferably, the patient is a human, however the present inventionextends to treatment and/or prophylaxis of other mammalian patientsincluding primates, livestock animals (e.g. sheep, pigs, cattle, horses,donkeys), laboratory test animals (e.g. mice, rabbits, rats, guineapigs), companion animals (e.g. dogs, cats) and captive wild animals. Inaccordance with the present invention, the agent is preferablyadministered to a patient by a parenteral route of administration.Parenteral administration includes any route of administration that isnot through the alimentary canal (that is, not enteral), includingadministration by injection, infusion and the like. Administration byinjection includes, by way of example, into a vein (intravenous), anartery (intraarterial), a muscle (intramuscular) and under the skin(subcutaneous). The agent may also be administered in a depot or slowrelease formulation, for example, subcutaneously, intradermally orintramuscularly, in a dosage which is sufficient to obtain the desiredpharmacological effect.

In another aspect, the present invention provides the use of an agentwhich blocks or inhibits IL-3 signalling events in, or in themanufacture of a medicament for, the treatment of a chronic inflammatorycondition in a patient.

In yet another aspect, the present invention provides an agent for thetreatment of a chronic inflammatory condition in a patient, wherein saidagent blocks or inhibits IL-3signalling events in the patient. In thisaspect of the invention, the agent as described above may be formulatedin a pharmaceutical composition together with one or morepharmaceutically acceptable excipients and/or diluents.

Compositions suitable for parenteral administration convenientlycomprise a sterile aqueous preparation of the active component which ispreferably isotonic with the blood of the recipient. This aqueouspreparation may be formulated according to known methods using suitabledispersing or wetting agents and suspending agents. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally-acceptable diluent or solvent,for example as a solution in polyethylene glycol and lactic acid. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, suitable carbohydrates (e.g. sucrose, maltose,trehalose, glucose) and isotonic sodium chloride solution. In addition,sterile, fixed oils are conveniently employed as a solvent or suspendingmedium. For this purpose, any bland fixed oil may be employed includingsynthetic mono- or di-glycerides. In addition, fatty acids such as oleicacid find use in the preparation of injectables.

The formulation of such therapeutic compositions is well known topersons skilled in this field. Suitable pharmaceutically acceptablecarriers and/or diluents include any and all conventional solvents,dispersion media, fillers, solid carriers, aqueous solutions, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like. The use of such media and agents forpharmaceutically active substances is well known in the art, and it isdescribed, by way of example, in Remington's Pharmaceutical Sciences,18th Edition, Mack Publishing Company, Pennsylvania, USA. Except insofaras any conventional media or agent is incompatible with the activeingredient, use thereof in the pharmaceutical compositions of thepresent invention is contemplated. Supplementary active ingredients canalso be incorporated into the compositions.

In a further aspect of the invention, there is provided a kit comprising(i) an agent as described above, and optionally (ii) instructions to usethe agent in accordance with a method for the treatment of a chronicinflammatory condition in a patient. The present invention is furtherillustrated by the following non-limiting Examples.

EXAMPLE 1

The effect of a neutralizing monoclonal antibody (mAb) to murine IL-3 ondisease progression was tested in the collagen-induced arthritis (CIA)model, the most widely used murine model for rheumatoid arthritis.

Male DBA/1 mice (8-12 weeks old, 10 mice per group) were immunizedintradermally with type II collagen in adjuvant on days 0 and 21 ¹⁸.Mice were assessed for redness and swelling of limbs and a clinicalscore was allocated for each limb using an established scoring system asfollows: 0—normal; 1—slight swelling and/or erythema; 2—extensiveswelling and/or erythema; 3—severe swelling; 4—severe swelling and/orrigidity. Severity of arthritis is expressed in terms of the meanclinical score totalled for all four limbs (range 0-16 per mouse). Micewere treated with 250 μg anti-IL-3 mAb (Southern Biotech)/mouse or PBSon day 21, 23, 25, 28 and 30. As can be seen in FIG. 1, there was asuppression of disease severity in the anti-IL-3-treated group.

This experiment is particularly encouraging as in this particularexperiment there was a very rapid induction of a very severe diseasewith the plateau in the control mice being reached quickly. This plateauis normally reached about one week later. The literature data for therelatively acute murine CIA indicates that neutralizing anti-IL-3 Ab isrequired soon after disease induction and is ineffective if delayed postdisease onset ¹⁹. This does not necessarily mean that IL-3 would not bea target in inflammation. We do not yet understand the backgroundinflammatory/autoimmune events responsible for driving diseases such asrheumatoid arthritis (RA) and how closely these events are mirrored inanimal models. Even for a chronic condition such as RA there arepatients with “acute onset” disease. Also, anti-IL-3 therapy might stillbe beneficial in RA, for example, since the disease often relapsesproviding opportunities to suppress its exacerbations. It should also beborne in mind that the commonly used anti-inflammatory glucocorticoidsare widely believed to act by down-regulating inflammatory mediator geneexpression at the transcriptional level—they do not work in vitro insurrogate inflammation assays if added subsequent to the incitingstimulus.

EXAMPLE 2

The role of IL-3 signalling as a pro-inflammatory cytokine in CD 14+monocytes was investigated. Peripheral Blood Mononuclear Cells (PBMC)were isolated from a Red Cross donor buffy pack. CD 14+ monocytes werethen purified by negative selection (MACS separation) such thatapproximately 80% of the cells were CD 14+ as assessed by flowcytometry. Monocytes were plated at a density of ˜1.8×10⁶ cells per 6 cmIWAKI (low adherence) TC dish and cultured for 7 days in RPMI+10% FCSand: IL-3 (3 ng/ml) alone, IL-3 (3 ng/ml) +IL-3-R antibody, IL-3 (0.3ng/ml) alone, or IL-3 (0.3 ng/ml)+IL-3-R antibody. IL-3-R antibody wasused at 1 ug/ml in each case. At day 4 new IL-3 and IL-3-R antibodieswere added. On day 7 cells were removed and counted before being lysedin RNA lysis buffer. The results indicate that IL-3 provides apro-survival stimulus for the monocytes in a dose dependent manner andthis effect was overcome by anti-IL-3R antibody (FIG. 2). Thus blockingor inhibiting IL-3 signalling in CD 14+ monocytes prevents theirsurvival and accumulation at sites of inflammation and as such providesa means to control inflammation.

EXAMPLE 3

Urticaria, commonly known as hives, is an inflammatory condition thatmanifests as recurrent wheals that range in size from severalcentimeters down to just a few millimeters. Generally the wheals arepink in color with a pale center and can be associated with a burning orprickly sensation. Urticaria can present at any age and 1%-5% of thepopulation will present with urticaria at some point in their lifetime.Chronic urticaria greatly impacts on quality of life, similar topatients with severe atopic dermatitis, psoriasis or acne. Most cases ofchronic urticaria are idiopathic in nature; however, it is becomingincreasingly clear that in many cases (35%-50%) auto-antibodies to thehigh affinity IgE receptor (FcεR1) or IgE itself are present, suggestingthat chronic urticaria may be an autoimmune disease. Mast cells andbasophils are the main cell types that express FcεR1 and respond toauto-antibodies in urticaria patients. When activated, mast cells andbasophils release large amounts of histamine, which is the main effectormolecule driving urticarial wheal formation. Both mast cells andbasophils express the IL-3 receptor and IL-3 can prime these cells toproduce increased levels of effector molecules, such as histamine, whenexposed to triggers such as IgE and C5a.

PBMC from seven urticaria patient samples and four normal donor sampleshave been analyzed. IL-3 can activate human basophils ex vivo (FIG. 3)and a neutralizing anti-IL-3Rα antibody (CSL360—see International PatentPublication No. WO2009/070844) can inhibit IL-3 induced human basophilactivation (FIG. 4). An ADCC optimized anti-IL-3Rα antibody (CSL362—anafucosylated variant of humanized and affinity matured anti-CD123 mAb168-26 as described in International Patent Publication No. WO2009/070844) can deplete human basophils from PBMC in a time dependentmanner (FIG. 5). Complete or near-complete basophil depletion wasobserved in three independent donors within 24 hours of CSL362 addition(FIG. 6). NK cell activation was observed within 24 hours of addition ofCSL362 suggesting that the depletion of basophils is via NK-cellmediated ADCC (FIG. 7).

EXAMPLE 4

This example demonstrates that anti-IL-3R antibodies can also affect thenumber and level of activation of basophils in vivo. Female BALB/c mice(10-12 weeks old) were treated with a tail vein intravenous injection ofanti-IL3Rα antibody 1C2 (see Example 5) or the control mouse IgG2a. Acommercial antibody Ba103 that targets against CD200R3 and has beenshown to specifically deplete mouse basophils²⁰, and its controlantibody rat IgG2b were tested alongside as the controls. All antibodieswere injected at 30 μg in 200 μl of PBS with the exception of 1C2 whichwas administered at 18 μg. A day later, peripheral blood and peritonealcells were isolated and single suspension of cells prepared. Cells werepre-incubated with anti-FcγRII-III to prevent nonspecific binding. Cellswere stained with FITC-conjugated anti-FcεRIα monoclonal antibody andPE-conjugated anti-CD49b monoclonal antibody to identify basophils(FcεRIα⁺ CD49b⁺). FITC-hamster IgG and PE-rat IgM were used as theisotype controls for FcεRIα and CD49b antibodies, respectively. Debriswas gated out using a forward scatter (FSC) versus side scatter (SSC)and dead cells were discriminated with 7-amino-actinomycin D (7-AAD)staining. Stained cells were then analysed with FACSCanto™ (BDBiosciences) and data analysed using FlowJo software.

A single intravenous injection of 18 μg anti-IL3Rα antibody 1C2 induceda drastic reduction in basophil frequency in peripheral blood, toapproximately 23% of the level in the isotype control (mouseIgG2a)-treated mice (FIG. 8). This depletion efficacy was comparable tothat from the administration of 30 μg Ba103. Unlike the effect onbasophils, no significant reduction in the frequency of peritoneal mastcells was noted in the mice treated with anti-IL3Rα or Ba103 antibodies(data not shown). The observation with Ba103 antibody on mast cells isconsistent with the studies by Obata and colleagues ²⁰.

EXAMPLE 5

1) Generation of murine IL3 receptor specific monoclonal antibodiesAntibody sequences that specifically recognised murine IL-3R alpha(mIL3Rα) were isolated from a library of human antibody sequencesexpressed as Fab fragments fused to the gIII protein on the surface ofthe filamentous bacteriophage M13 (Dyax Corp.). Anti-mIL3Rαphage-displayed antibody fragments were isolated by incubation of thephage library with a commercially-available purified recombinant fusionprotein consisting of amino acids 17-331 of mIL3α fused to residues100-330 of human IgG1 (Fc fragment) with a short polypeptide linker(sequence: IEGRID) supplied by R&D systems Inc.

Specifically-bound phage were enriched and isolated as individual clonesusing standard methods. Individual clones were tested for specificbinding to both the original target (mIL3Rα-Fc fusion) and to theextracellular domain of mIL3Rα expressed as residues 1-331 with aC-terminal hexa-histidine tag (msIL-3R-6His). Phage clones that bound tothese targets and not to control proteins were selected for furtheranalysis. Unique clones were identified by DNA sequencing of bothpolypeptide chains of the encoded antibody and the binding affinity ofthese clones for mIL-3R-6His was quantified by competitive ELISA. Cloneswith acceptable affinity were selected for re-engineering and expressionas chimeric antibodies (human variable regions and murine IgG2a/kappaconstant regions) for further analysis.

2) Mammalian expression vector construction for transient expressionHeavy and light chain variable regions for the mIL3Rα-specificantibodies were PCR amplified from the phagemid vectors using standardmolecular biology techniques. The heavy chain variable region was thencloned into the mammalian expression vector pcDNA3.1(+)-mIgG2a, which isbased on the pcDNA3.1(+) expression vector (Invitrogen) modified toinclude the murine IgG2a constant region and a terminal stop codon. Thelight chain variable region was cloned into the expression vectorpcDNA3.1(+)-mx, which is based on the pcDNA3.1(+) expression vectormodified to include the murine kappa constant region. The expressionvectors also contained a Kozak translation initiation sequence, an ATGstart codon and appropriate signal peptides.

3) Cell Culture

Serum-free suspension adapted 293-T cells were obtained from GenechoiceInc. Cells were cultured in FreeStyle™ Expression Medium (Invitrogen)supplemented with penicillin/streptomycin/fungizone reagent(Invitrogen). Prior to transfection the cells were maintained at 37° C.in humidified incubators with an atmosphere of 8% CO2.

4) Transient Transfection

Transient transfection of the anti-mIL3Rα expression plasmids using293-T cells was performed using 293fectin transfection reagent(Invitrogen) according to the manufacturer's instructions. The light andheavy chain expression vectors were combined and co-transfected with the293-T cells. Cells (1000 ml) were transfected at a final concentrationof 1×106 viable cells/ml and incubated in a Cellbag 2L (Wave Biotech/GEHealthcare) for 5 days at 37° C. with an atmosphere of 8% CO2 on a 2/10Wave Bioreactor system 2/10 or 20/50 (Wave Biotech/GE Healthcare). Theculture conditions were 35 rocks per minute with an angle of 8°.Pluronic® F-68 (Invitrogen), to a final concentration of 0.1% v/v, wasadded 4 hours post-transfection. 24 hours post-transfection the cellcultures were supplemented with Tryptone N1 (Organotechnie, France) to afinal concentration of 0.5% v/v. The cell culture supernatants wereharvested by centrifugation at 2500 rpm and were then passed through a0.45 μM filter (Nalgene) prior to purification.

5) Analysis of Protein Expression

After 5 days 241 of culture supernatant was electrophoresed on a 4-20%Tris-Glycine SDS polyacrylamide gel and the antibody was visualised bystaining with Coomassie Blue reagent.

6) Antibody Purification

Anti-mIL3Rα antibodies were purified using protein A affinitychromatography at 4° C., where MabSelect resin (5 ml, GE Healthcare, UK)was packed into a 30 ml Poly-Prep empty column (Bio-Rad, CA). The resinwas first washed with 10 column volumes of pyrogen free GIBCO DistilledWater (Invitrogen, CA) to remove storage ethanol and then equilibratedwith 5 column volumes of pyrogen free phosphate buffered saline (PBS)(GIBCO PBS, Invitrogen, CA). The filtered conditioned cell culture media(1L) was loaded onto the resin by gravity feed. The resin was thenwashed with 5 column volumes of pyrogen free PBS to remove non-specificproteins. The bound antibody was eluted with 2 column volumes of 0.1Mglycine pH 2.8 (Sigma, Mo.) into a fraction containing 0.2 columnvolumes of 2M Tris-HCl pH 8.0 (Sigma, Mo.) to neutralise the low pH. Theeluted antibody was dialysed for 18 hrs at 4° C. in a 12 mlSlide-A-Lyzer cassette MW cutoff 3.5 kD (Pierce, Ill.) against 5L PBS.The antibody concentration was determined by measuring the absorbance at280 nm using an Ultraspec 3000 (GE Healthcare, UK) spectrophotometer.The purity of the antibody was analysed by SDS-PAGE, were 2 μg proteinin reducing Sample Buffer (Invitrogen, CA) was loaded onto a Novex10-20% Tris Glycine Gel (Invitrogen, CA) and a constant voltage of 150Vwas applied for 90 minutes in an XCell SureLock Mini-Cell (Invitrogen,CA) with Tris Glycine SDS running buffer before being visualised usingCoomassie Stain, as per the manufacturer's instructions.

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1-38. (canceled)
 39. A method for the treatment of a chronicinflammatory condition in a patient in need thereof, which comprisesadministration to the patient of an antibody which selectively binds toIL-3R alpha and wherein the antibody has a modified Fc region to provideenhanced effector function.
 40. The method according to claim 39 whereinthe chronic inflammatory condition is urticarial
 41. The methodaccording to claim 39 wherein the enhanced effector function is selectedfrom the group consisting of enhanced binding to Fc receptors, enhancedADCC activity, enhanced ADCP activity and enhanced CDC activity.
 42. Themethod according to claim 39 wherein the antibody has enhanced ADCCactivity.
 44. The method according to claim 42 wherein the antibody isafucosylated.
 45. The method according to claim 39 wherein the urticariais chronic urticaria.